Daniel Branton

Lamellar and hexagonal lipid phases visualized by freeze-etching

Abstract 1. 1. Rapid freezing of lipid-water preparations preserves the structure of the high temperature phases. Both lamellar and hexagonal phases can be readily demonstrated by electron microscopy and X-ray observations. 2. 2. Without careful controls, contaminants are readily deposited on fresh fracture faces. The contamination can take the form of particles resembling those found on natural membranes. 3. 3. Neither degree of saturation, degree of hydration, nor cholesterol admixture significantly affects the appearance of lamellar fracture faces which appear uniformly smooth. On uncontaminated specimens, no structures were found which resembled the particulate material of natural membranes.

Subunits in chloroplast lamellae.

The structure of chlorophyll-containing lamellae has been studied in freeze-etched preparations of isolated and in situ chloroplasts. These lamellae are composed of at least two classes of subunits. The first type of subunit has an average diameter of 175 A and is not less than 90 A high. It corresponds in size and distribution to the quantasome. The second type of subunit has an average diameter of 110 A and appears to form part of an embedding matrix around the larger, 175 A, units. Thus, chloroplast lamellae consist of a matrix within which are densely packed subunits that form the major structural constituents of the photosynthetic membrane.

Fracture Planes in an Ice-Bilayer Model Membrane System

Experiments with transferred stearate layers were performed to determine the location of fracture planes in frozen ice-lipid systems. Bilayers and multilayers of carbon-14-labeled stearate were frozen in contact with an aqueous phase and then fractured. The distribution of radioactivity on both sides of the fracture showed that the stearate layers were cleaved apart predominantly in the plane of their hydrocarbon tails. Because bilayers split in this manner, it was possible to measure time-dependent exchange of label between the layers. Exchange occurred with a half-time of 50 minutes in the presence of calcium and 25 minutes in the absence of calcium. Since stearate bilayers and multilayers are models of hydrophobically stabilized structures, the strong influence of their hydrophobic region on the fracture plane provides an explanation of how the freeze-etch technique of electron microscopy can expose inner, hydrophobic faces of cell membranes.

Fracture faces in frozen outer segments from the guinea pig retina

SummaryOuter segments from the retina of the guinea pig have been examined with the freeze-etch technique. In many ways their appearance corroborates previous descriptions of their fine structure. However, the fracture faces of the disc membrane are distinctive and unlike those of any other membrane examined by freeze-etching. One face has the appearance of shallow, irregularly shaped pits surrounded by steep, interconnecting ridges. The other face has the appearance of worn cobblestone pavement. The “stones” are somewhat irregularly shaped, are tightly packed together, and have dimensions of 200–250 Å. In transverse fracture, a single disc membrane is represented by a pair of ridges and has a thickness of about 90 Å.Evidence is presented that the disc membranes split during fracture and that the two faces seen in freeze-etched replicas are apposed in the intact membrane. This interpretation, assuming fractures split membranes, is compared with one assuming fractures occur along membrane surfaces. The inadequacies of both interpretations are discussed. Plastic deformation can occur during fracture. Such deformation may explain some interpretational difficulties and may account for the lack of perfect match between the two fracture faces in the disc membrane.

Lipid- and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes

Abstract The lipids in cell membranes of Acholeplasma laidlawii were enriched with different fatty acids selected to produce membranes showing molecular motion discontinuities at temperatures between 10 and 35 °C. Molecular motion in these membranes was probed by ESR after labelling with 12-nitroxide stearate, and structure in these membranes was examined by electron microscopy after freeze-etching. Freeze-etching and electron microscopy showed that under certain conditions the particles in the A. laidlawii membranes aggregated, resulting in particle-rich and particle-depleted regions in the cell membrane. Depending upon the lipid content of the membrane, this aggregation could begin at temperatures well above the ESR-determined discontinuity. Aggregation increased with decreasing temperature but was completed at or near the discontinuity. However, cell membranes grown and maintained well below their ESR-determined discontinuity did not show maximum particle aggregation until after they had been exposed to temperatures at or above the discontinuity. The results show that temperatures at or near a phase transition temperature can induce aggregation of the membrane particles. This suggests that temperature-induced changes in the lipid phase of a biological membrane can induce phase separations which affect the topography of associated proteins.

Organization of mitochondrial structure as revealed by freeze-etching

Abstract 1. 1. A freeze-etch study of rat-liver and rabbit-heart mitochondria reveals detailed structure not recognized by the usual procedures of electron microscopy. 2. 2. These include a fibrous network in the matrix of contracted mitochondria, particulate components associated with the inner membrane, and smooth patches covering the particles of the inner membrane. 3. 3. Chemical fixation is found to prevent maximum resolution of fine detail but glutaraldehyde-fixed mitochondria still maintain the general morphological features observed by other techniques.

Subliming Ice Surfaces: Freeze-Etch Electron Microscopy

Vacuum sublimation of oriented single crystals of ice at temperatures from -110 to -60 degrees Celsius was studied by electron microscopy with the freeze-etch technique. Sublimation etches the ice surface to produce pits and asperities and above -85 degrees Celsius causes extreme surface roughening. The etch pits are ascribed to surface dislocations, and the extreme roughening is ascribed to the departure from unity of the vaporization coefficient. The asperities could not be attributed to impurities; they may be related to the whiskers that others have observed at higher temperatures.

Gas vacuole development in a blue-green alga.

De novo productioni of gas vacutoles can be induced in the blue-green alga Nostoc muscorum by transferring the cells from a defined medium to distilled water. The unusual ultrastructure of the gas vacuole membranes permits their easy recognition when specimens are prepared for electron microscopy by freeze-etching. The youngest gas vacuoles are biconical organelles; 48 hours after induction the gas vacuoles reach their miaximum observed length when they are cylinders (1.5 by 0.1 �) with cornical ends.

Dry, High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.

The correlation between the saturation of membrane fatty acids and the presence of membrane fracture faces after osmium fixation

Abstract In osmium-fixed membranes, there is a decrease in the membrane fracture faces concomitant with an increase in unsaturation of the membrane fatty acids. The data suggest that both the number of double bonds and their position in the fatty-acid chain are critical to the disappearance of membrane fracture faces.